This project is devoted to forward genetic screens for new mutants that affect trafficking of Notch, and potentially other &amp;#947;-secretase substrates, through intracellular membrane compartments. Although screens for mutants affecting cell proliferation, polarity, and patterning have led to the identification of genes influencing endosomal Notch trafficking, systematic screens for mutations that directly alter the cellular distribution of Notch have not been performed. Based on our previous analysis of the effects of Ca2+-ATPase mutations on the trafficking of Notch through the secretory pathway, the highly polarized structure of Drosophila disc cells, and the genetic techniques available for producing clones of lethal mutations in developing fly tissues, we reason that Drosophila is a suitable model for attempting this type of cell biological screen. To perform the screen, we mutagenize male flies, establish balanced stocks, induce tissue clones that are homozygous for a single mutagenized chromosome arm, and analyze the distribution of Notch protein in the mutant clone relative to adjacent wildtype tissue. At present, we are completing our screen of the second chromosome. Approximately 4000 mutagenized chromosome arms have been screened, resulting in the recovery of 91 mutations, which define over 20 separate genes (each represented by 2-8 mutant alleles). The different mutants can be classified with respect to their effects on Notch trafficking and the intracellular membrane compartment affected. Further characterization of several of these new mutants, including mapping and molecular cloning, is now underway.